This invention relates to a method for the covalent modification of tissue with small molecules and functional proteins.
The importance of interactions between proliferating cells and substrate surfaces, and especially surface proteins and carbohydrates, has long been an active area of study. It is increasingly well understood how surface molecular composition, topology, and geometric patterning of extracellular matrix (“ECM”) molecules influence specific biological processes and have a critical role in cell growth regulation and differentiation, cell migration, apoptosis, and general morphogenesis. Accordingly, there has been significant effort expended to develop methods to create surfaces for tissue culture that are patterned with specific biologically relevant molecules in order in influence the aforementioned properties.
A variety of methods are known for the chemoselective modification of specific functional groups found in complex proteins. In general, this chemistry is solution chemistry, and these methods have been developed for the modification of soluble proteins. The electrophilic aromatic substitution of the tyrosine phenol functionality is a very specific example. Often, chloramine-T is used to reduce iodide, resulting in the introduction of iodine (most often a radioactive isotope such as I-129 or I-131) into the protein's tyrosine residues at the position ortho to the phenol. However, this chemistry is limited, as the conjugation of complex molecules to the target protein is often the goal.
For a more general example, the amine residues found in most proteins (at the N-terminus as well as from lysine residues) are readily modified, most commonly with electrophilic acylating agents. While this chemical modification is straightforward and relatively general, there are often numerous amines that exhibit similar reactivity in a given protein, and this methodology is considered to be rather non-selective. Additionally, this strategy precludes the use of amine-containing functionalities in the acylating agent, therefore dramatically limiting the structural complexity of the modification reagent.
A functional group that has proven extremely useful for site-specific protein modification is the free sulfhydryl. This functionality can be either found in native protein (for example in albumin), revealed via the reduction of disulfides bonds found in the native protein (for example, the reduction of F(ab)2 IgG antibody fragments to release Fab-SH fragments), or introduced into the protein by reaction with a reagent such as Traut's reagent. Protein-bound thiol groups can be selectively alkylated using maleimide reagents or α-halo carbonyl compounds (although this is typically less chemoselective, as α-halocarbonyls also react with protein amines at a competitive rate). Since free sulfhydryls are rarely found in native protein, the number of reducible disulfides is usually very limited, and the maleimide alkylation of thiols is very chemoselective, this chemistry is considered to be more precise and controllable than the amine alkylation methodology. Additionally, the chemoselectivity of this methodology allows the use of highly functionalized conjugation reagents.
Many additional methods for the modification of soluble proteins have been developed but have been applied much less widely. For example, carbonyl residues generated by the oxidation of sugar residues found in glycosylated proteins can be reacted with amines (reductive alkylation) or hydrazide-containing reagents (to afford hydrazones). However, at this time there have been no reports of producing patterns of functional proteins on fresh tissue surfaces.